Expression of neuronal NO synthase α- and β-isoforms in skeletal muscle of mice

Knowledge of the primary structure of neuronal NO synthase (nNOS) in skeletal muscle is still conflicting and needs further clarification. To elucidate the expression patterns of nNOS isoforms at both mRNA and protein level, systematic reverse transcription (RT)-PCR and epitope mapping by qualitative immunoblot analysis on skeletal muscle of C57/BL6 mice were performed. The ability of the nNOS isoforms to form aggregates was characterized by native low-temperature polyacrylamide electrophoresis (LT-PAGE). The molecular analysis was focused on the rectus femoris (RF) muscle, a skeletal muscle with a nearly balanced ratio of nNOS α- and β-isoforms. RT-PCR amplificates from RF muscles showed exclusive exon-1d mRNA expression, either with or without exon-μ. Epitope mapping demonstrated the simultaneous expression of the nNOS splice variants α/μ, α/non-μ, β/μ and β/non-μ. Furthermore, immunoblotting suggests that the transition between nNOS α- and β-isoforms lies within exon-3. In LT-PAGE, three protein nNOS associated aggregates were detected in homogenates of RF muscle and tibialis anterior muscle: a 320 kDa band containing nNOS α-isoforms, while 250 and 300 kDa bands consist of nNOS β-isoforms that form homodimers or heterodimers with non-nNOS proteins.